PB1792: NGS VS PCR FOR THE DETECTION OF NPM1 GENE MUTATIONS IN ACUTE MYELOID LEUKEMIA.
Authors: N. Severina, Y. Sidorova, N. Risinskaya, B. Biderman, A. Pshenychnyy, N. Ryzhikova, I. Lukianova, A. Kashlakova, A. Sudarikov
Journal: HemaSphere (2022)

Background:NPM1 gene mutations are found in 30% of acute myeloid leukemia (AML) cases. It is a reliable marker for the evaluation of therapy outcome and minimal residual disease (MRD) monitoring. The most often mutations are 4bp insertions in exon 12, affecting 2 functionally important tryptophan residues (W288 and W290) in the C-terminal region of nucleophosmin and causing a frameshift. As a result, a leucine-rich motif (L-xxx-V-xx-V-x-L) is formed, which is a signal sequence for protein export from the nucleus to the cytoplasm. Commonly NPM1 gene mutations are detected by fragment analysis (FA) of PCR fragment of 12 exon for the presence of 4bp insert. Alternatively, allele-specific polymerase chain reaction (AS-PCR) to detect inserts belonging to the A, B, or D type (representing up to 90% of all inserts) is used. However, about 10% remaining cases are non-standard inserts and intron polymorphisms that require more precise evaluation. Aims: Evaluation of diverse approaches for efficient NPM1 gene mutation analysis and their robustness for the diagnostics. Methods: The study included 80 DNA samples from AML patients admitted to the National Research Center for Hematology in 2019-2021 that were positive for NPM1 gene mutations detected by FA performed with the Nanophor-5 genetic analyzer (Synthol, Russia). The type of insert was assessed by AC-PCR. Samples in doubt were evaluated by targeted next-generation sequencing (NGS) on MySeq (Illumina, USA). Results: In 62 out of 80 samples, the type of insert (A, B, or D) was reliably determined by AS-PCR. In 9 cases type of insert was not detected. In further 9 cases, amplification ΔCt between wild-type and mutant allele was out of reliable range. These 18 samples were examined by NGS, and 2 out of 18 showed an intron deletion, which, presumably, does not affect the function of the protein, but leads to the appearance of an additional peak on FA. In 16 out of 18 samples, non-standard inserts were detected (Table 1). Type A inserts were found in 48 out of 78 cases (61.5%), type B - in 2 out of 78 (4.5%), and type D - 15.4% (12 out of 78). We have detected atypical inserts in 16 out of 78 cases (20.5%), which is more than reported in the literature. 14 out of 16 atypical inserts result in a leucine-rich motif, as do standard inserts. In 4 out of 16 cases, one of the 2 functionally important tryptophan residues was retained (W280), therefore possibly conserving nuclear localization signal. The effect of partial preservation of the nucleolar localization of nucleophosmin on prognosis in AML has not been studied yet. Image:Summary/Conclusion: FA is still a golden standard for screening NPM1 gene mutations in AML. AS-PCR could be effectively used for monitoring cases with standard inserts. However, NGS greatly facilitates non-standard inserts identification, appropriate allele-specific primers design for MRD analysis, and avoiding false-positive FA results for cases with intron localization of the insert.